Immunoglobulins are glycoproteins that exist in serum and tissue/body fluid of all mammals. Immunoglobulins have a function of recognizing foreign antigens. Immunoglobulins are involved in biological defense through activation of their effector functions. Effector functions involve activation of the complement system, which is induced by antibody-antigen binding, and effects of promoting cell phagocytosis, antibody-dependent cellular cytotoxicity, mediator release, and antigen presentation Fc receptors (FcR) that exist on the cell surface.
There are 5 different classes of human immunoglobulins, namely IgG, IgA, IgM, IgD, and IgE. IgG can be classified into the 4 subclasses of IgG1, IgG2, IgG3, and IgG4. Also, IgA can be classified into the 2 subclasses of IgA1 and IgA2. The basic structure of immunoglobulin is made up of 2 homologous L chains (light chains) and 2 homologous H chains (heavy chains). The immunoglobulin class and subclass are determined by H chains.
Different immunoglobulins are known to have different functions. For instance, the level of complement-binding ability is ranked in the following order: IgM>IgG3>IgG1>IgG2. Also, the level of affinity to FcγRI (Fc receptor I) is ranked in the following order: IgG3>IgG1>IgG4>IgG2. In addition, IgG1, IgG2, and IgG4 can bind to protein A.
Human antibodies that have been collected and purified from blood are used as pharmaceutical agents. In recent years, many monoclonal antibodies have been subjected to clinical tests. Also, many such antibodies have been placed on the market. However, most of the monoclonal antibodies used as pharmaceutical agents that have been placed on the market or clinically developed are derived from IgG1. Very few such antibodies are derived from IgG4. It has been known that IgG4 is characterized by having lower levels of complement activation and antibody-dependent cellular cytotoxicity than IgG1. Thus, it is considered that IgG4-derived products are appropriate for use as pharmaceutical agents having such characteristics. Upon activation of the effector function of an antibody that is bound to an antigen, a cell expressing the antigen is damaged via a complement system or another cell. In a case in which such antigen is a cancer-specific antigen or the like, the function of cell damage is very important in terms of medicinal properties. However, in the cases of some antigens, such function might cause adverse effects. An example of such antigen is CD40. The CD40 gene plays an important role in immune control. Anti-CD40 antibodies that inhibit binding between a CD40 ligand and CD40 are considered to have high potential as pharmaceutical agents. However, CD40 expression is found in various other types of cells besides immunocytes. Therefore, if the effector function of such an antibody causes damage to a CD40-expressing cell, there is a possibility that severe adverse effects could be induced. Thus, IgG4 is considered to be a preferable antibody against such aforementioned antigens.
Meanwhile, in order to produce IgG4 that serves as a pharmaceutical agent, certain problems must be considered. Such problems involve the existence of an IgG4 half antibody having no SS bond between its H chains in vivo. (An antibody generally comprises 2 L chains and 2 H chains; however, a half antibody comprises a single L chain and a single H chain.) Also, problematic is the production of a half antibody in a case in which IgG4 is expressed in animal cells such as CHO for the production of a pharmaceutical agent. Such half-antibody formation is a serious problem that is a cause for concern in terms of the production of pharmaceutical agents. However, it has been reported that half-antibody formation can be inhibited by introducing a single amino acid substitution into a constant region (see Non-Patent Document 1). In addition, although IgG4 is known to have a lower level of antibody-dependent cellular cytotoxicity compared with IgG1, IgG4 has FcR-binding ability, although at a weak level. However, such FcR-binding ability can be further attenuated by introducing a mutation into a hinge region of IgG4 so as to improve IgG4 (see Patent Documents 1 to 3).
In accordance with the methods described above, it becomes possible to produce almost ideal IgG4. However, in terms of production of a pharmaceutical agent comprising IgG4, there are still problems to be overcome. Such problems involve instability of IgG4 antibodies at low pH. In general, a method of affinity purification with the use of protein A is used to produce antibodies as pharmaceutical agents. In such case, a low-pH buffer is often used for elution of antibodies each binding to protein A. In addition, in order to remove viruses, it is desired that antibodies be treated at low pH for a certain period of time. However, as shown in the present invention, it has been known that IgG4 antibodies are likely to form an aggregate since they have poor stability at low pH compared with IgG1 antibodies. It has been reported that incorporation of an aggregate into a pharmaceutical agent causes promotion of infusion reactions, complement activation, or antibody-dependent cellular cytotoxicity. Thus, it is easily assumed that such reactions and the like give rise to adverse effects. Therefore, it is very important to reduce the amount of aggregate as less as possible.    [Patent Document 1] JP Patent No. 3101690    [Patent Document 2] U.S. Pat. No. 5,648,260    [Patent Document 3] U.S. Pat. No. 5,624,821    [Non-Patent Document 1] S. Angal et al., Molecular Immunology 1993, 30, 105-108